1. Field of Invention
The present invention relates to a fusion gene found in squamous cell carcinomas, detection of the rearrangement and monoclonal antibodies specific for SCCA1, SCCA1/A2, SCCA2/A1 and SCCA2.
2. Description of the Prior Art
Squamous cell carcinoma antigen (SCCA) is a serological marker for squamous cell carcinomas (SCC) of the uterine cervix, lung, head and neck, vulva, and esophagus (1, 2). It was originally purified from the TA-4 complex from human cervical squamous cell carcinoma, with a molecular weight of 42-48 kDa (1, 3). The antigen consists of more than 10 proteins and iso-electric focusing of the antigen reveals two subfractions, an acidic (pI<6.25) and a neutral (pI≧6.25) isoform (4). The difference in molecular weight is probably due to modification (5).
Cloning of the cDNA of SCCA shows that it belongs to the family of serine protease inhibitors (serpins) (6). Further cloning of the genomic region on chromosome 18q21.3 reveals two tandemly arrayed genes (7). The more telomeric one, the original SCCA, was designated SCCA1, whereas the more centromeric one was designated SCCA2 (FIG. 1A). They both contain eight exons and the putative intron-exon boundaries, splice sites, initiation codons, and terminal codons are identical. They are 98% identical at the nucleotide level (FIG. 2) and 92% identical at the amino acid level (FIG. 3). The deduced pI value shows that the neutral isoform is coded by SCCA1, and the acidic isoform by SCCA2. Alternatively spliced variant mRNA from both the genes have been found resulting in proteins 52 and 21 amino acids shorter (5).
In humans the serpins map to one of two chromosomal clusters. PI6, PI9 and ELNAH2 map to 6p25, whereas PI8, Bomapin, PAI2, SCCA1, SCCA2, Headpin and Maspin map to 18q21.3 (FIG. 1A) (7-12). These clusters are supposed to have arisen via two independent interchromosomal duplications and several rounds of intrachromosomal duplications (9). The chromosome region 18q has often been reported as a region with high frequency of rearrangements (9, 13-16). The targets and functions of serpins are not fully understood. For most, the primary functions are regulation of proteolytic events associated with coagulation, fibrinolysis, apoptosis and inflammation, but alternative functions such as hormone transport and blood pressure regulation have been reported (17-24).
Although SCCA1 and SCCA2 are nearly identical they differ in their reactive site loops (FIGS. 2 and 3). SCCA1 inhibits the papain-like cystein proteinases cathepsin S, K, and L (25, 26) while SCCA2 inhibits the chymotrypsin-like serine proteinases cathepsin G and mast cell chymase (27). Studies of the reactive site loop (RSL) of SCCA1 show that the RSL is essential for cystein proteinase inhibition (28). The variable portion of the RSL dictates the specificity of the target proteinases shown by RSL swap mutants of SCCA1 and SCCA2 and single mutants (28, 29). It is likely that serpins utilize a common RSL-dependent mechanism to inhibit both serine and cystein proteinases.
The biological role of SCCA1 and SCCA2 are not fully understood. They are considered to be inhibitory serpins. Data suggest that SCCA1 is involved in apoptosis and expression makes cancer cells resistant to several killing mechanisms by inhibition of apoptosis (30). The role of SCCA2 expression in cancer cells is still unclear. In normal tissue SCCA antigen may have some specific role during epidermal maturation (5).
Recent studies using discriminatory monoclonal antibodies and polymerase chain reaction (PCR) have shown that both SCCA1 and SCCA2 are expressed in the suprabasal layers of the stratified squamous epithelium of the tongue, tonsil, esophagus, uterine cervix and vagina, Hassall's corpuscles of the thymus, some area of the skin and in the stratified columnar epithelium of the conducting airways (31). In squamous cell carcinomas of the lung and head and neck, SCCA1 and SCCA2 were co-expressed in moderately and well-differentiated tumors. In contrast to previous studies using nondiscriminatory antibodies, these data show that there were no differential expression between SCCA1 and SCCA2 in normal and malignant tissue. Previous results have shown that SCCA2 was only detected at the peripheral parts of the tumor (32). This discrepancy may be due to differences between immunohistochemical techniques and antibody specificities (31). It has been reported that false positive results may often be caused by contamination with saliva or sweat during assay procedure (1). Cataltepe et al. suggest that the SCCAs in saliva are derived from the squamous epithelial cells lining mucosal surfaces of the upper digestive tract (31).
Normally, SCCA1 and SCCA2 are detected in the cytoplasm of squamous epithelial cells (31), but not in the circulation (33). The antigen, which appears in the serum of patients with SCC, may be a function of SCCA-over-production by tumor cells and their normal turn over (34). It has been reported that the SCCA detected in serum by using antibody radioimmuno-assay or RT-PCR is mainly SCCA2 (1, 35, 36) but other studies using PCR indicate that both antigens can be amplified and detected in patient samples (37).
Serum concentrations present in patients with SCC are correlated to the clinical stage and to the degree of histological differentiation of the tumor (1). For cervical cancer several studies show a correlation between the pretreatment values and the clinical outcome (1, 38-43). Studies also show a correlation between high SCCA levels and tumor volume. Recurrence or progressive disease could be detected several months before clinical evidence (39). Similar results are seen for squamous cell carcinomas of the lung, vulva, head and neck and esophagus (1, 2, 44, and 45). In all these studies, they have measured the total SCCA level. Recently a new sELISA was developed using discriminating antibodies for SCCA1 and SCCA2 (33).